PROJECT SUMMARY/ABSTRACT The entry of human immunodeficiency virus (HIV-1) into host cells is mediated by the envelope glycoprotein (Env) trimer ((gp120/gp41)3). As the only virus-specific molecule on the viral surface, Env is the major target for host neutralizing antibodies. During virus entry, binding to the receptors triggers conformational changes in the metastable Env that allow the fusion of viral and target cell membranes. Env conformational flexibility, which is important for virus entry, also contributes to HIV-1's ability to evade the host antibody response. Prior to receptor engagement, the HIV-1 Env trimer on virions can potentially sample three conformations: a metastable closed conformation (State 1), the open CD4-bound conformation (State 3), and an intermediate partially open conformation (State 2). Primary HIV-1 Envs are maintained in State 1 by multiple intramolecular and intersubunit interactions, rendering these Envs relatively resistant to the binding of potentially neutralizing antibodies. CD4 binding drives Env from State 1 to State 2 and then into State 3, the prehairpin intermediate. The conformational flexibility of HIV-1 Env trimers has created challenges for structural studies and for the design of vaccines. Stabilization of soluble gp140 (sgp140) Env trimers has been achieved by introduction of an SOS disulfide bond linking gp120 and gp41, substitution of proline for isoleucine 559, and truncation of the gp41 ectodomain at residue 664. Crystal and cryo-EM structures of these sgp140 SOSIP.664 trimers have been solved. A cryo-EM structure of a cytoplasmic tail-deleted Env trimer (Env?CT) in complex with the PGT151 neutralizing antibody was virtually identical to the sgp140 SOSIP.664 structure. Although these HIV-1 Env trimer structures are widely believed to represent the closed State-1 conformation, recent data from single- molecule Fluorescence Resonance Energy Transfer (smFRET) and crosslinking/mass spectrometry analyses indicate that this assumption is incorrect. In fact, the gp120 subunits of the sgp140 SOSIP.664 trimers and the Env?CT/PGT151 complex are both in a State 2-like conformation! Crosslinking/mass spectrometry of the native cell-surface Env and sgp140 SOSIP.664 trimers revealed differences in the conformation of several gp120 and gp41 regions! The surprising results described above indicate that substantial differences exist between the conformations of the native State-1 Env and the structurally well-characterized sgp140 SOSIP.664 and PGT151-bound Env?CT trimers, which apparently represent a default intermediate (State 2-like) conformation. Thus, we currently lack detailed information about the structure of the State-1 HIV-1 Env trimer, the major target for neutralizing antibodies and virus entry inhibitors. The proposed work seeks to devise approaches to express and purify HIV- 1 Env trimers enriched in a State-1 conformation. Given the importance of shape to the humoral immune system, we will then test the hypothesis that preparations of State 1-enriched immunogens will elicit primary HIV-1- neutralizing antibodies more efficiently than Envs not specifically enriched for a State-1 conformation.